So-called “prion diseases” are a serious social problem. Examples of prion diseases include Transmissible Spongiform Encephalopathies (TSEs) such as Scrapie, Bovine Spongiform Encephalopathy (BSE), and Creutzfeldt-Jakob Disease (CJD).
Based on various evidences, it has become clear that such prion diseases are caused by infectious prion protein (pathogenic isoform of prion protein) (PrPsc).
In order to prevent transmission of prion diseases of humans and animals and ensure the safety of drugs and foods, various attempts have been made to detect infectious prion protein (pathogenic isoform of prion protein) contained in samples.
However, humans and animals originally have, in their bodies, noninfectious prion protein (normal prion protein) (PrPc) not causing prion diseases. Surprisingly, normal prion protein has the same amino acid sequence (primary structure) as pathogenic isoform of prion protein, and the only difference between normal and pathogenic iso form of prion proteins having the same amino acid sequence is in their higher-order structures.
In general, in a case of detecting two kinds of proteins distinctively from each other, a specific antibody that can distinguish between these two kinds of proteins can be used. However, a specific antibody that can distinguish between pathogenic isoform of prion protein and normal prion protein has not yet been obtained probably due to their identical amino acid sequence described above. That is, there is still no possibility of practical use of a method for detecting infectious prion protein (pathogenic isoform of prion protein) contained in a sample using a specific antibody.
Under the circumstances, a method for detecting pathogenic isoform of prion protein is limited mainly to the following two types.
One is a method in which a sample suspected to contain pathogenic isoform of prion protein (infectious prion protein) is injected into the brains of test animals and the test animals are bred over a long period of time to monitor neuropathological changes in brain specimens from the test animals.
This method is reliable, but unfortunately, requires too much time and money. Therefore, this method is used only to calibrate other various detection methods, and has not reached routine use.
The other one is a method using protease K. It is known that normal prion protein is easily degraded by (i.e., sensitive to) protease K, but on the other hand, pathogenic isoform of prion protein is hard to be degraded by (i.e., resistant to) protease K probably due to their higher-order structures. Therefore, pathogenic isoform of prion protein can be detected by utilizing a difference in sensitivity (resistance) to degradation by protease K between normal and pathogenic isoform of prion proteins. For example, a sample is treated and not treated with protease K, and is then analyzed by, for example, immunoblotting using a polyclonal antibody. In a case where protein bands are detected by immunoblotting of the sample treated with protease K at the same positions as those detected by immunoblotting of the sample not treated with protease K, it is judged that the protein bands represent pathogenic isoform of prion protein. On the other hand, in a case where protein bands detected by immunoblotting of the sample not treated with protease K disappear (i.e., not detected) by immunoblotting of the sample treated with protease K, it is judged that the protein bands represent normal prion protein.
This detection method using protease K is now widely used, and many variations thereof have been proposed (see, for example, Patent Documents 1 to 3).
However, this method necessarily involves enzymatic treatment, and therefore requires time to perform the enzymatic reaction and is complicated in that it is necessary to create conditions suitable for the enzymatic reaction. Therefore, in principle, this method has drawbacks in that it is poor in quickness and simplicity and that a relatively expensive enzyme is absolutely necessary as a reagent.
Patent Document 1: Japanese Patent Application Laid-open No. 10-267928
Patent Document 2: Japanese Patent Application Laid-open No. 11-32795
Patent Document 3: Japanese Patent Application Laid-open No. 2003-121448